Molecular characterization of <i>Spirometra</i> isolates across the USA

<i>Spirometra</i> is a genus of zoonotic cestodes with an ambiguous species-level taxonomic history. Previously, <i>Spirometra mansonoides</i> was considered the only species present in North America. However, recent molecular data revealed the presence of at least three distinct species in the USA: <i>Spirometra</i> sp. 2 and 3, and <i>Spirometra mansoni</i>. This study aimed to elucidate the diversity and potential host associations of <i>Spirometra</i> species among companion animals in the USA. Samples (<i>N</i> = 302) were examined from at least 13 host species, including mammals, amphibians and reptiles. Sample types included eggs isolated from faeces (<i>n</i> = 222), adult specimens (<i>n</i> = 71) and plerocercoids (<i>n</i> = 9) from 18 different states and 2 territories across the USA. Extracted genomic DNA was subjected to PCR targeting a fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. Generated sequences (<i>n</i> = 136) were included in a phylogenetic analysis. <i>Spirometra mansoni</i> was detected in domestic cats (<i>n</i> = 76), dogs (<i>n</i> = 12), a White's tree frog (<i>n</i> = 1), a Cuban knight anole (<i>n</i> = 1), a green iguana (<i>n</i> = 1) and a serval (<i>n</i> = 1) across 15 states and Puerto Rico. <i>Spirometra</i> sp. 2 was found only in dogs (<i>n</i> = 3) from Florida and <i>Spirometra</i> sp. 3 was found only in cats (<i>n</i> = 41) from 17 states. All plerocercoid samples were consistent with <i>S. mansoni</i>. The results confirm that at least three distinct <i>Spirometra</i> species are present and established in companion animals, such as dogs and cats, and likely are using various native and exotic species as paratenic hosts within the USA.